HIV-1 infection is influenced by several host factors, including components of DNA repair pathways. One example is Rad18, a member of the post-replication DNA repair system and also an important element in the maintenance of genome stability. Rad18 interacts with and stabilizes HIV-1 integrase. Moreover, Rad18 has a negative influence on HIV-1 infection, an effect exerted at a step between reverse transcription and integration. Rad18 also has a negative effect on infection by MLV, and adenovirus, suggesting that double stranded DNA is the ultimate target of the inhibitory activity. We hypothesize that HIV-1 integrase serves as a bridge to allow Rad18 to access viral cDNA, where it might recruit other effectors from DNA repair pathways, leading to cDNA degradation. We propose to determine whether Rad18 interaction with integrase is responsible for the inhibition of HIV-1 infection by Rad18. This will be accomplished by assessing the difference of cDNA synthesis between wild type viruses and viruses lacking integrase, in Rad18-/- and Rad18+/+ cells. Furthermore we plan to map the domains and residues that participate in the association of the two molecules. Mutation of the integrase residues involved in the interaction with Rad18 will yield protein variants that cannot associate with Rad18, and therefore viruses that are not inhibited by Rad18. Conversely, Rad18 mutants that cannot interact with integrase will not have an inhibitory effect on HIV-1 infection, in contrast to what observed with wild-type Rad18. We will evaluate all mutants in the context of viral infection. Furthermore, we will also examine the effect of overexpression of the interacting domains by themselves on viral infection, to assess whether they can suppress the Rad18 inhibition by titration of the host protein (in the case of integrase domains) or by competition with cellular Rad18 (in the case of Rad18 domains). We also aim to investigate the functional conservation, in HIV-1 infection, of human homologues of yeast genome stability factors that inhibit Ty retrotransposition. We will initially analyze two genes, FEN-1 and WRN, whose orthologs in yeast are known to influence Ty1 activity. Knockdown cell lines will be obtained using shRNA methodology. These cell lines will be tested for HIV-1 infection, and we will characterize which phase of the viral life cycle is affected. The proposed research will elucidate the role of Rad18 in inhibiting HIV-1 infection and reveal new host factors that modulate viral infection. The results will open perspectives for new anti-retroviral therapy development. 7. Project Narrative. The goal of this proposal is to elucidate the mechanism of HIV-1 inhibition by DNA repair molecule Rad18 and identify novel cellular suppressors of HIV-1, by taking advantage of their potential conservation from yeast to man. The identification and understanding of these proteins may lead to the ability to harness multiple levels of inhibition against which viruses would unlikely have the capacity to adapt. This can then translate not only in the precious expansion of our knowledge about the viral biology but possibly in the discovery of amenable anti- retroviral targets.